Abstract One important yet understudied aspect in nanomaterial applications is the cell biology of nanomaterial transport into cells and through tissues. To overcome the cell membranes and reach their sites of action within, nanomaterials are often complexed with targeting ligands, such as cell-penetrating peptides (CPPs), to engage with cell surface receptors. The receptor-mediated cell entry is also advantageous in concentrating more nanomaterials into certain cell or tissue types (e.g. tumors). However, the cellular machineries that mediate and/or regulate the cargo transport after receptor binding remain to be further elucidated. In this proposal, I aim to decode the cellular machinery of one such cell entry pathway, macropinocytosis (MP). MP has relatively large endocytic vacuoles (>200 nm in diameter) and thus can more readily engulf cargo as large as nanoparticles. However, MP pathway, especially receptor-mediated ones, has remained elusive due to the lack of specific tracers. Recent studies on CPPs, including ours, have shed new light in this problem by discovering two novel receptor-mediated MP processes, which are initiated by peptide ligation with either heparan sulfate (HS) proteoglycans or another cell surface protein, neuropilin-1 (NRP1). We further showed that NRP1 and HS pathways are two functionally distinct subtypes of receptor-mediated MP, and generated peptide tools to exclusively trace one or the other of these two pathways. In the Aim 1, I will perform a genome-wide RNAi screening to identify genes and signaling cascades that play essential roles in initiating and regulating receptor-mediated MP. Silver-based nanoparticles coated with NRP1- or HS-binding peptides will be used to probe NRP1 and HS pathways simultaneously. In the Aim 2, I will determine the regulation of NRP1- and HS-mediated nanomaterial delivery by a variety of Ras oncogenes, which are among the most commonly mutated genes in human cancers. My proposed studies hold great potential for unveiling important cellular machineries for nanomaterial transport, boosting the efficiency of intracellular delivery, and opening the door for personalized nanomaterial delivery and even nanomedicine.